The long-term goal of this project is to define the structural parameters of glycosphingolipids that promote formation of membrane domains and to determine the impact of glycosphingolipid membrane organization on the functional regulation of a protein that catalyzes the intermembrane transfer of glycolipids, i.e. glycolipid transfer protein. The ability of glycosphingolipids to organize into domains in biological membranes is postulated to be a key feature, not only in their own intracellular sorting and trafficking, but also in the sorting and trafficking of proteins with glycosylphosphatidylinositol covalent anchors. The processes by which glycosphingolipid-enriched domains are formed and maintained are not well understood and may involve specific proteins that can bind and transfer glycosphingolipids between membrane surfaces. By using model membrane systems that provide distinct yet complementary information about lipid-lipid and lipid- protein interactions, the following specific aims will be addressed: (1) to determine the structural features of glycolipids that modulate their mixing interactions with phospholipids and sterols, and to define the physical nature of the lamellar environment that is produced by glycolipid-lipid interactions using monolayer and calorimetric approaches; (2) to ascertain the role that glycolipid lateral organization plays in regulating the activity of glycolipid transfer protein by using monolayer and fluorescence techniques; and (3) to overexpress glycolipid transfer protein using molecular biological approaches and provide a foundation for future investigations of site- directed mutagenized forms of glycolipid transfer protein. Achieving these aims will be of fundamental importance in developing ways to control glycosphingolipid availability and accessibility at the cell surface. Such manipulations could be key to preventing infection by certain bacteria (N. gonorrhea, pathogenic E. coli, and cholera) and viruses (rotaviruses and HIV) as well as lead to better ways to target drugs to the surfaces of tumor cells that express oncogenically-related glycosphingolipids.